The clinical symptoms of patients with autoimmune haemolytic anaemia (AIHA) may range from no symptoms at all to severe haemolysis with life-threatening anaemia. The aim of the present study was to characterize the autoantibodies serologically and to evaluate their activity in vitro in monocyte monolayer assay (MMA) in consecutive direct antiglobulin test (DAT)-positive patients from a haematological department. We also aimed to evaluate the monocyte production of cytokines in vitro and relate all findings to in vivo haemolysis. Twenty-nine patients with positive DAT were included in the study and clinical characteristics were documented. The patients were divided into three groups based on laboratory parameters, severe haemolysis, moderate haemolysis and no sign of haemolysis. Severe haemolysis was related to a strongly positive DAT and positive results in MMA. When eluates were analysed in MMA, a positive result was associated with a higher concentration in the supernatants of interleukin (IL)-8 after 6 h incubation. We conclude that MMA activity and in vitro cytokine production may reflect the in vivo activity of autoantibodies. This may be of importance in understanding the mechanisms of haemolysis and in predicting the harmfulness of antibodies, especially when blood transfusions are needed.